High efficiency tissue specific compound delivery system using streptavidin-protein a fusion protein

ABSTRACT

The present invention relates to methods and compositions that can be employed to introduce toxins and nucleic acids into the cytoplasm or nucleus of a eukaryotic cell, particularly a cell of a higher vertebrate. The invention particularly concerns the use of a fusion protein of streptavidin and protein A sequences to form a non-covalent complex of a toxin or nucleic acid and an antibody.

[0001] This is a division of application Ser. No. 08/566,421, filed Nov.30, 1995, now U.S. Pat. No. 6,497,881. This prior application is herebyincorporated herein by reference, in its entirety.

INTRODUCTION

[0002] The present invention relates to methods and compositions thatcan be employed to introduce toxins and nucleic acids into the cytoplasmor nucleus of a eukaryotic cell, particularly a cell of a highervertebrate. The invention allows for the efficient and specific deliveryof the toxins and nucleic acids into cells that bind an antibody. Theinvention particularly concerns the use of a fusion protein ofstreptavidin and protein A sequences to form a non-covalent complex of atoxin or nucleic acid and an antibody.

[0003] The invention provides a method of treatment of human disease bythe introduction of toxins or antisense nucleotides into human cells,e.g., tumor cells, in vivo or ex vivo. The invention also providesmethods of conducting biological research and methods useful in theproduction of biological products by introducing exogenous duplex DNAmolecules into cultured cells.

BACKGROUND OF THE INVENTION

[0004] The selective introduction of compounds into the cytoplasm ornucleus of specific cells has been a valuable technique in biologicaland medical research and in medical practice. Cell specific targeting ofcytotoxins has been accomplished by complexing toxins with cell bindingproteins that can preferentially bind targeted cells. The cell-bindingproteins of the complex can be either antibodies, particularlymonoclonal antibodies, or protein ligands, e.g.,/growth factors/whichrecognize the corresponding surface antigens or receptor. Complexes oftoxin and antibody have been termed immunotoxins.

[0005] Conventionally, the toxin and cell binding protein of the complexhave been linked covalently through either chemical coupling or genefusion. Conventional immunotoxins have been made by chemically linking atoxin component to an antibody, typically a monoclonal antibody, with aheterobifunctional cross-linking reagent that is non-specific.Accordingly, this method yields a heterogeneous product in which sometoxin molecules block the antibody's ability to bind antigen by linkingto the F(ab) portion of the antibody. Additionally, the couplingchemistry can partially destroy the toxins activity.

[0006] Many of the problems associated with chemical conjugation havebeen overcome through the generation of single-chain fusion toxins usingrecombinant DNA technology. However, this technology requires that a newrecombinant toxin for each target cell. The biological activity of eachnew recombinant toxin is unpredictable.

[0007] Alternative techniques have been developed in which the cytotoxinis non-covalently linked to the antibody or ligand. One such techniqueexploits the specific interaction between Staphylococcal aureus proteinA and immunoglobulins to generate antibody complexes with twospecificities. According to this technique, protein A is complexed withantibodies of two different specificities: a toxin specific antibody anda cell surface specific antibody. Such complexes have been used todeliver ricin toxin into targeted cells (Laky, et al., 1986/1987,Immunology Letters 14:127-132). In a second immunotoxin targetingsystem, single chain antibodies are fused with streptavidin which has astrong and specific binding affinity for biotin. Using this construct,biotinylated toxin was delivered into a target cell (Dubel, et al.,1995, Journal of Immunological Methods, 178:201-209).

[0008] Recently, Sano et al. described a fusion protein consisting ofstreptavidin and one or two immunoglobulin G (IgG)-binding domains ofprotein A in Escherichia coli. (U.S. Pat. No. 5,328,985, issued Jul. 12,1994, which is hereby incorporated by reference in its entirety). Thestreptavidin-protein A (ST-PA) fusion protein has functional biotin andIgG binding sites. Sano further described complexes of thestreptavidin-protein A fusion protein, a monoclonal antibody to BSA, andbiotinylated horseradish peroxidase.

[0009] Sano also described a method of labeling cells using the ST-PAfusion protein. Cells were incubated with an antibody to a cell surfaceantigen, Thy-1. The chimeric protein-biotinylated marker complex wassubsequently added to the cell suspension. This technique was used todeliver biotinylated FITC to the surface of cells having Thy-1 antigenon their surface. However, Sano did not describe or suggest the use ofthe ST-PA fusion protein to deliver compounds into the cytoplasm ornucleus of specific cells.

[0010] Immunotoxins appear to enter the cell via receptor-mediatedendocytosis (Pastan et al., 1986, Cell 47:1-44 and Pirker et al., 1987,Lymphokines 14:361-382). Binding of the antibody moiety of theimmunotoxin complex to the surface receptor is followed by, first,clustering of the complex into coated pits and then by internalizationof the complex into endosomes or receptosomes within the cell(Middlebrook et al., 1994, Microbiol. Rev., 48:199-221; Morris et al.,1985, Infect. Immun. 50:721-727; Fitzgerald et al., 1980, Cell21:867-873). During the journey into the cell, the complex may betransported through different intracellular compartments that vary in pHand proteolytic enzyme activity before the toxins are translocatedacross an intracellular membrane and into the cell cytoplasm where theycan cause cell death.

[0011] A second area which has been developed concerns methods forintroducing nucleic acids into cells. The most widely used methodsemploy calcium phosphate or DEAE-dextran to promote uptake of nucleicacids. These methods appear to involve the steps of DNA attachment tothe cell surface, entry into the cytoplasm by endocytosis, andsubsequent transfer into the nucleus. Maniatis, Laboratory CloningManual, volume 2, 16.30. Depending upon the cell type, up to 20% of apopulation of cultured cells can take up DNA using calcium phosphate orDEAF-dextran.

[0012] Electroporation is an alternative transfection method in which anelectric field is applied to open pores in the cell plasma membrane. DNAappears to enter the cell through these pores.

[0013] Liposomes have also been used to introduce nucleic acids intocells. According to this technique, artificial lipid-bilayer vesiclescontaining cationic and neutral lipids mediate the transfer of DNA orRNA into cells. The mechanism of liposome-mediated transfection, is notwell understood, but it appears that negatively charged phosphate groupson DNA bind to the positively charged surface of the liposome, and thatthe residual positive charge binds to negatively charged sialic acidresidues on the cell surface.

[0014] Sano did not use the complex to introduce nucleic acid into thecell. Sano et al. described DNA-antibody complexes with the ST-PA fusionprotein by incorporating a single biotin molecule at one end of alinearized pUC 19 plasmid. In contrast to the methods of transfectingnucleic acids into the cell which are described above.

SUMMARY OF THE INVENTION

[0015] The invention relates to a method of delivering toxins or nucleicacids into specific cell types and to the complexes for the practice ofthe method. According to the invention, an antibody that recognizes acell surface antigen is non-covalently bound to the antibody bindingsite of a ST-PA fusion protein; a biotinylated toxin or nucleic acid isbound to the biotin-binding site. In an alternative embodiment, thetoxin or nucleic acid can be bound to a third biotinylated molecule, anadapter, which is bound to the biotin binding-site.

[0016] In one embodiment of the present invention, a nucleic acid isdelivered into a specific cell type. The nucleic acid can be abiotinylated single stranded nucleic acid bound to the biotin bindingsite of the ST-PA fusion protein. In an alternative embodiment, thenucleic acid can be a duplex nucleic acid that forms a complementarytriplex with a biotinylated single stranded nucleic acid, which is inturn bound to the biotin binding site.

[0017] The method of the invention relates to the steps of forming acomplex between a streptavidin-protein A fusion protein; an antibody,that is specific for a cell surface protein, which undergoes endocytosisafter binding with the antibody; and some targeted material e.g. abiotinylated multidrug resistance (mdr) gene product, prodrug, toxin ornucleic acid; isolating the complex from toxin that is not bound to thebiotin binding site; and exposing the target cell, to the complex sothat the targeted material enters the cell.

Definitions

[0018] As used herein, toxin, refers to holotoxins, modified toxins,catalytic subunits of toxins, or any enzymes not normally present in acell that under defined conditions cause the cell's death. Abiotinylated toxin refers to a toxin that is either directlybiotinylated or one that is bound to a biotinylated adapter.

[0019] The term “antibody” refers to any molecule which contains one ormore functional antigen binding domains and an Fc domain thatspecifically binds protein A.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1A. Schematic representation of immunotoxin or recombinanttoxin binding to the corresponding surface antigen or receptor on thecell surface.

[0021]FIG. 1B. Schematic representation of the streptavidin-proteinA/biotinylated macromolecule complex binding to the correspondingsurface antigen or receptor on the cell surface.

[0022] FIGS. 2A-C. Comparison of the delivery of biotin-β galactosidaseinto A431 cells of an ST-PA/anti-EGFR mAB/biotin-β galactosidase complex(B) and ST-TGF/biotin-β-galactosidase complex (C).

[0023]FIG. 3. Time course analysis of cell β-galactosidase stainingafter transfer of β-galactosidase into the cells.

[0024]FIG. 4A. Cell lines and cell surface molecules targeted by theST-PA-biotin-βgalactosidase-antibody complex.

[0025] FIGS. 4B-G. Transfer of ST-PA/mAB/biotin-β galactosidase intohuman cells Daudi (B and E), HL-60 (C and F) and KG1 (D and G), wherethe mAB is specific for HLA-DR, CD33, or CD34 cell surface molecules.

[0026] FIGS. 5A-C. Schematic of the pAT-β galactosidase expressionvector construct (A) for forming triplex DNA with biotinylated poly (dT)oligonucleotide (B-C).

[0027]FIG. 6A. Amino acid homology between fibronectin (SEQ ID NO:1) andstreptavidin (SEQ ID NO:2). Bold type indicates homologous residues. TheRGD and RYD domain of each protein is underlined. The sequence shown forfibronectin begins at residue 1481, and the streptavidin sequence beginsat residue 49.

[0028]FIG. 6B. Oligonucleotide primers DES (SEQ ID NO:3) and DER (SEQ IDNO:4) designed to modify the RYD sequence of the streptavidin gene.

[0029]FIG. 6C. Schematic of the methodology to be used in modifying theRYD sequence of streptavidin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention provides a method of specific delivery oftargeted material, e.g., toxins, prodrugs, mdr gene products, or nucleicacids into cells by a complex of the targeted material, an antibodyspecific for a cell surface antigen on the cell, which antigen isendocytosed when the cell is exposed to an effective concentration ofthe antibody, and a ST-PA fusion protein.

[0031] The streptavidin-protein A fusion protein (ST-PA) binds,non-covalently the cell specific antibody in the antibody binding siteand the biotinylated targeted material in the biotin binding site. Themanner and method by which the targeted material is biotinylated is notcritical; the invention includes the use of any and all such methods andreagents.

[0032] In one embodiment, the invention provides a method for thespecific destruction of cells, e.g., the destruction of tumor cells in ahost or ex vivo in short term or long term culture. A toxin can beselected from the group consisting of: thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. The toxin is biotinylated and thecomplex of PA-ST/Ab/biotinylated toxin is formed. An effective amount ofthe complex can then be administered to the host or to the ex vivoculture system.

[0033] In an alternative embodiment the invention provides a method ofusing an antibody to a cell to introduce into the cell a cytotoxicprodrug, i.e., a non-toxic compound that is converted by an enzyme,normally present in the cell, into a cytotoxic compound. The embodimentcontemplates the use of a complex of the ST-PA chimeric protein with atumor selective monoclonal antibody and prodrug. Compounds suitable asprodrugs include by way of example glutamyl derivative of a benzoic acidmustard alkylating agent, phosphate derivatives of etoposide ormitomycin C, and phenoxyacetamide derivative of doxorubicin.

[0034] A further embodiment of the invention contemplates a complex forthe delivery of single stranded nucleic acid into cells. As used herein,the term “single stranded nucleic acid” refers to both naturally andnon-naturally occurring nucleic acids. The nucleic acid to be deliveredcan be biotinylated using any of the methods currently available, suchas, random incorporation, extension reactions using DNA polymerase, andPCR with biotinylated primers. Such nucleic acids can be used tospecifically destroy mRNAs to which they are complementary.

[0035] A further embodiment of the invention concerns the introductioninto a cell of a duplex DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed. The directincorporation of biotin into a duplex DNA can interfere with bothreplication and transcription of the DNA. To overcome these problems,the duplex DNA to be delivered into the cell is modified to include asequence that can form a region of triplex nucleic acid with a singlestranded nucleic acid.

[0036] Triplex DNA formations have been described and typically consistof T-A-T and CG-C nucleotide triads. The third strand of the triplexoccupies the major groove of an A-form DNA helix and forms Hoogsteenbase pairs with the homopolymeric duplex DNA. Alternatively, a triplexcan be formed with duplex DNA. As used herein, the term “triplex” refersto a structure formed by Hoogsteen base pairing between a duplex DNA anda single stranded nucleic acid.

[0037] In this embodiment, the complex contains a duplex DNA Hoogsteenpaired to a single stranded nucleic acid, which is biotinylated andcomplexed with the ST-PA/mAb complex. The duplex DNA can be a linearduplex DNA, which is suitable for recombination into the genome of acell or, alternatively, the duplex can be a circular or supercoiledcircular DNA, which can episomally replicate.

[0038] This embodiment of invention can be used under any circumstancesit is desired to introduce cloned DNA into a cell, e.g., to express aproduct or to alter the phenotype of the cell, to investigate thefunction of any cloned gene.

[0039] A further embodiment of the invention comprises a complex of anantibody to a cell surface protein found on an antigen presenting cell,the ST-PA fusion protein, and a biotinylated protein of a pathologicalbacteria or virus, such a complex can be used to localize the antigen toantigen presenting cells and thereby enhance the immune response of CD4positive T cells relative to that of other lymphocytes.

[0040] The complexes of the present invention can be formed by simplyadmixing ST-PA, a monoclonal antibody, and the biotinylated material inthe appropriate ratios. The components can be mixed in any order.

[0041] The ST-PA fusion protein forms tetramers which bind up to fourbiotinylated molecules and four IgG molecules, which are each bivalent.Without limitation as to theory, the octovalent binding of complexes ofthe invention to the cell surface is believed to cause the complexes tohave superior binding and internalization properties compared to otherimmunotoxins and immunopharmaceutical complexes.

[0042] Biotin-blocked streptavidin is capable of specificallyinteracting with cell surfaces through an Arg-Tyr-Asp sequence presentin the protein (the “RYD site”). This site is distinct from thebiotin-binding cleft of the protein and bears high homology to theRGD-containing cell binding domain of fibronectin which mediatesfibronectin-cell surface interactions (Alon et al, 1993, Europ. J. CellBiol. 60:11). Studies have suggested that streptavidin acts as a closemimetic of fibronectin (Alon et al, 1993, Europ. J. Cell Biol. 60:1-11).

[0043] The conserved RYD and RGD domains of fibronectin and streptavidinfunction as universal recognition sequences for interactions with manymembrane-bound receptors. In one embodiment of the invention, thestreptavidin component of the complex can be modified to alter the RYDsite. As used herein, the term “modified RYD sequence” includes anyalteration to the RYD site or flanking region which eliminates thenon-biotin binding site-related interaction of streptavidin with cellsurface proteins. One such modification is the replacement of asparticacid by glutamic acid.

EXAMPLES

[0044] 1.1 Materials and Methods

[0045] Plasmid, pTSAPA-2, described in U.S. Pat. No. 5,328,985, issuedJul. 12, 1994, carries the chimeric gene of streptavidin and protein A(region E and D). Expression and purification of the gene fusion ofST-PA was carried out according to the methods that follow.

[0046] Fusion-Protein-Preparation:

[0047] Bacterial Strain lysogen BL21 (DE3) (pLysS) was transformed withthe pTSAPA-2 streptavidin-protein A fusion expression vector. Thetransformed strain was grown at 37° C. in LB media supplemented with 50μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. When theabsorbance at 600 nm of the culture was between 0.8 and 1.0 OD, 100 mMisopropyl β-D-thiogalactopoyranoside (IPTG) dissolved in water was addedto a final concentration of 0.4 mM to induce the T7 RNA polymerase geneplaced under the lac UV5 promoter. After the induction, the cells wereincubated at 37° C. with shaking for 2 hours.

[0048] Purification of streptavidin-protein A fusion chimeric proteinwas carried out at 4° C. or on ice unless otherwise indicated. Theculture (100 ml) of BL21 (DE3) (pLysS)(pTSAPA-2) incubated for 2 hoursafter the induction was centrifuged at 2,900×g for 15 min. The cellpellet was suspended in 10 ml of 2 mM EDTA, 30 mM Tris-Cl (pH 8.0), 0.1%Triton X-100, 0.5 mM PMSF to lyse the cells and the lysate was stored at−70° C. until used. To the thawed cell lysate, PMSF, leupeptin, andpepstatin A were added to final concentrations of 0.5 mM, 1 μM, and 1μM, respectively. The lysate was then treated with 10 μg/ml ofdeoxyribonuclease 1 and 1 0 μg/ml ribonuclease A in the presence of 12mM MgSO₄ at room temperature for 20 minutes. The mixture was centrifugedat 39,000×g for 15 minutes and the pellet was dissolved in 100 ml of 7 Mguanidine hydrochloride overnight at 4° C. with stirring. After thepellet was dissolved the protein was then dialyzed against 150 mM NaCl,50 mM Tris-Cl (pH 7.5), 0.05% Tween 20, 0.1 mM PMSF, 1 μM leupeptin, 1μM pepstatin A, 0.02% NaN₃ To achieve slow removal of the guanidinehydrochloride, the dialysis bag containing the protein solution was leftovernight in the dialysis solution (˜1,000 ml) without stirring,followed by 3 changes of the dialysis solution and dialysis withstirring at 4° C. The dialysate was centrifuged at 39,000×g for 15minutes, and the supernatant was applied to an IgG Sepharose 6 Fast Flowcolumn (1.2×1.1 cm) previously washed with 5-10 bed volumes of TSTBuffer. The column was then equilibrated with 2-3 bed volumes ofeach: 1) 0.5 M Acetic acid, pH 3.4 (pH adjusted with NH₄CH₃COOH(NH₄Ac);2) 150 mM NaCl, 50 mM Tris-Cl (pH 7.5), 0.05% Tween 20 (TST Buffer); 3)0.5 M Acetic Acid, pH 3.4; and 4) TST. The sample was applied to thecolumn and the unbound protein was removed by washing the columnwith: 1) 10 bed volumes of TST, and 2) 2 bed volumes of 5 mM NH₄Ac, pH5.0. Elution was performed with 0.5 M Acetic Acid, pH 3.4. The eluatewas collected in 1-2 ml fractions, and the fractions having the greatestOD at 280 were dialyzed against 1 M NaCl, 50 mM sodium carbonate (pH11.0). The dialysate was clarified by centrifugation at 39,000×g for 15minutes, and applied to a 2-iminobiotin agarose column (1.2×1.2 c.m)previously equilibrated with 1 M NaCl, 50 mM sodium carbonate (pH 11.0).After the unbound proteins were removed with the same solution, thebound proteins were eluted with 6 M urea, 50 mM ammonium acetate (pH4.0). The eluted proteins were dialyzed against Tris-buffered saline[TBS; 150 mM NaCl, 20 mM Tris-C1 (pH 7.5)] containing 0.02% NaN₃, andthe dialysate was stored at 4° C. after filtration through a 0.22 μmfilter (Millex-GV, Millipore).

[0049] Formation of the ST-PAS/mAb/biotinylated β-Galactosidase Complex:

[0050] A mixture of 2 μg of antibody, ˜28 μg of fusion protein, and ˜2units of biotinylated β-Galactosidase was incubated at room temperaturefor at least 10 minutes. After this incubation, the complex is ready touse.

[0051] β-Galactosidase Staining: (X-GAL staining)

[0052] β-Galactosidase staining of cells that adhere to the plate wasperformed according to Sanes, et al., 1986, EMBO J. 5: 3133-3142.

[0053] The protocol of Molecular Probes, Inc. was used to detect lacZβ-Galactosidase gene expression in cells that grow in suspension.

[0054] The above protocols were used to determine if the complex thatcontains the antibody coupled to the fusion protein and the biotinylatedβ-Galactosidase enzyme was successfully transduced into the cell ofchoice. If the transduction was complete, the cells were blue afterovernight incubation.

[0055] 2) Modification of Streptavidin Protein RYD Sequence.

[0056] Biotin-blocked streptavidin binds specifically (Kd=3×10⁸M) tocell surfaces, presumably via an RYD containing sequence that isdistinct from the biotin-binding cleft of the protein.

[0057] Alternation of the RYD domain (sequence) to other amino acidresidues is expected to eliminate the non-biotin related specificsurface binding of a large variety of cells.

[0058] One way to modify the RYD sequence would be to change the RYDsequence to RYE. The change of RYD sequence to RYE can be achieved byintroducing a point mutation using sequential PCR steps. This can beachieved by designing two primers, DES (SEQ ID NO: 3) and DER (SEQ IDNO: 4) (see FIGS. 6A-C) and using the pTSAPA-2 expression vector as atemplate of the streptavidin gene. First, PCR is carried out using twopairs of primers: T7 promoter/DER and T7 terminator/DES. Second, the twoamplified DNA fragments are then purified and pooled into one sample. Asecond round of PCR is then performed using the pooled purified productsof the first round as templates and the T7 promoter and T7 terminatorprimers. The mutated RYE sequence in the streptavidin gene component isconfirmed through sequence analysis of the product of the second roundof PCR.

[0059] 3) Delivery of Biotin-β-galactosidase into A431 Cells.

[0060] In order to study the ability of ST-PA to deliver compounds intoa cell, the delivery of this complex was compared with that of a complexin which core streptavidin was covalently linked to the TGFα receptor.

[0061] pTSA-TGFα, an expression vector for streptavidin-TGF-α (ST-TGF)was constructed by replacing the gene of protein A in pTSAPA-2 with amature human TGF-α gene (amino acids 1-50).

[0062] To demonstrate the capability of ST-PA and ST-TGFα fusionproteins to deliver biotinylated protein into specific cells types,biotinylated β-galactosidase was complexed with the streptavidincomponent of the fusion protein. Delivery of β-galactosidase into A431cells was quantitated using known staining techniques and FACS analysis.

[0063] ST-PA/biotin β-galactosidase was complexed with anti-EGFR mAb andthe resulting complex was then incubated with A431 human epidermoidcells over-expressing epidermal growth factor receptor (EGFR).Alternatively, ST-TGF was mixed with biotin-β-galactosidase and theresulting complex was administered to A431 cells.

[0064] As shown in FIG. 2B, ST-PA fusion protein efficiently deliveredbiotin-β-galactosidase into A431 cells through EGFR on its surface(positive cells >99%) (see FIG. 2C). The ST-TGFα fusion protein alsodisplayed efficient delivery of biotin-β-galactosidase into A431 cells(positive cells >99%). Surprisingly, the amount ofbiotin-β-galactosidase delivered into each cell by the ST-TGFα fusionprotein was lower than that observed in the ST-PA delivery system (themean fluorescence activity observed was 214 and 2402, respectively).

[0065] The highly efficient delivery by ST-PA fusion protein ofbiotin-β-galactosidase into A431 cells may be due to the four bindingsites for biotin and IgG contained on each ST-PA tetramer (FIG. 1B). Ina time course experiment shown in FIG. 3, more than 99% of cellsdemonstrated positive staining for β-galactosidase up to 2 days aftertransfer of biotin-β-galactosidase into the cell.

[0066] 4) Delivery of Biotin β-galactosidase into Cells Using mAbs ofDifferent Specificity.

[0067] The experimental procedure applied in studying the delivery ofbiotin β-galactosidase into A431 cells was also used to study thedelivery of the antibody/ST-PA/biotinylated β-galactosidase complex intoother cell types. The study utilized antibodies that recognize severaldifferent cell surface molecules. The cell lines and cell surfacemolecules used in this experiment are summarized in FIG. 4A. As shown inFIGS. 4B-G, the ST-PA/mAb complex was highly efficient (positivecells >99%) in transferring β-galactosidase into the human cell typestested by way of the HLA-DR, CD33 and CD34 molecules present on thesurface of these cells.

[0068] 5) pAT-β-galactosidase Expression Vector.

[0069] Although biotin can be incorporated into DNA (e.g. expressionplasmids), random incorporation of biotin into DNA may result in a lossof transcriptional activity. In order to retain the transcriptionalactivity of the DNA to be introduced into the cell, a new gene transfersystem was developed which utilizes a pAT-expression vector having apoly(dA)/poly(dT) tract downstream of the expression cassette (FIG. 5A).According to this transfer system, the DNA to be delivered into the cellis cloned into the expression vector. The pAT-expression vector formstriplex DNA with a biotinylated poly(dT) oligonucleotide (SEQ ID NOS: 5and 6) which binds to the biotin binding site of the ST-PA fusionprotein (FIGS. 5B-C). The antibody bound to the antibody binding site ofthe ST-PA fusion protein targets the cells into which the DNA is to bedelivered.

[0070] The protocol for triplex DNA formation and DNA transfer of theβ-galactosidase gene into cells is as follows. The β-galactosidase geneis cloned into the expression cassette of the pAT-expression vector(FIG. 5A). The mixture of 4 μg of pAT-β-galactosidase expression vectorand 20 pmol of Biotinylated poly(dT) oligonucleotide (Promega) in TMNbuffer (10 mM Tris, pH 8.0, 10mM MgCl₂, 50 mMNaCl), is incubated at 37°C. for 1 hour. ST-PA fusion protein (0.5 μg) and mAB (1.0 μg) are thenadded to the mixture and the resulting admixture is incubated at roomtemperature for 30 minutes. The triplex DNA-ST-PA-mAb complex is addedto the cells (1×10⁶) and incubated at 37° C. for 48 hours.β-galactosidase activity is detected by FACS.

REFERENCES

[0071] Sano, T. and Cantor, C. R. 1991. A streptavidin-protein A chimerathat allows one-step production of a variety of specific antibodyconjugates. Bio/Technol. 9:1378-1381.

[0072] Pastan, I. and FitzGerald, D. 1991. Recombinant toxins for cancertreatment. Science. 254:1173-1177.

[0073] Goshom, S. C., Svensson, H. P., Kerr, D. E., Somerville, J. E.,Senter, P. D. and Fell, H. P. 1993. Genetic construction, expression,and characterization of a single chain anticarcinoma antibody fused tob-lactamase. Cancer Res. 53:2123-2127.

[0074] Siegall, C. B., Xu, Y.-H., Chaudhary, V. K., Adhya, S.,FitzGerald, D. and Pastan, I. 1989. Cytotoxic activities of a fusionprotein comprised of TGFα and pseudomonas exotoxin. FASEB J.3:2647-2652.

[0075] Ghetie, M.-A., Laky, M., Moraru, T. and Ghetie, V. 1986. ProteinA vectorized toxins-I. Preparation and properties of protein A-Ricintoxin conjugates. Mol. Immunol. 23:13731379.

[0076] Kiyama, R., Nishikawa, N. and Oishi, M. 1994. Enrichment of humanDNAs that flank poly(dA). poly(dT) tract by triplex DNA formation. J.Mol. Biol. 237:193-200.

[0077] Ito, T., Smith, C. L. and Cantor, C. R. 1992. Sequence-specificDNA purification by triplex affinity capture. Proc. Natl. Acad. Sci. USA89:495-498.

[0078] Alon R., E. Bayer, M. Wilchek, 1993, Cell Adhesion tostreptavidin via RGD-dependent integrins, European Journal of CellBiology 60: 1-11.

We claim:
 1. A complex for transferring a compound into a cell, whichcomprises: (a) a streptavidin-protein A fusion protein having anantibody binding site and a biotin binding site; (b) an antibody, boundto the antibody binding site, which antibody is specific for a cellsurface protein, and which cell surface protein undergoes endocytosisafter binding with the antibody; and (c) a biotinylated compound, boundto the biotin binding site.
 2. The complex of claim 1 in which thebiotinylated compound is selected from the group consisting ofbiotinylated multidrug resistance (MDR) gene product, biotinylatedsingle stranded nucleic acid, double stranded DNA that forms triplexstructure with a biotinylated single stranded nucleic acid having ahomopurine or homopyrimidine portion, and biotinylated protein of apathological bacteria or virus.
 3. The complex of claim 1 in which thereare four antibody binding sites and four biotin binding sites.
 4. Thecomplex of claim 1 in which the streptavidin component of saidstreptavidin-protein A fusion protein has a modified RYD sequence. 5.The complex of claim 1 which further comprises an antibody that bindstransferrin receptor.
 6. The complex of claim 1, wherein the antibodyrecognizes a surface antigen selected from the group consisting of: (a)human lymphocyte antigen (HLA-DR); (b) cluster of differentiation(CD33); (c) cluster of differentiation (CD34); and (d) epidermal growthfactor (EGF) receptor.
 7. The complex of claim 1 in which the antibodyis an IgG antibody.
 8. A pharmaceutical composition, comprising: (a) thecomplex of claim 1, and (b) a pharmaceutically acceptable carrier, whichcomposition is substantially free of biotinylated compound not bound tothe streptavidin-protein A fusion protein.
 9. A complex for transferringa prodrug into a cell, which comprises: (a) a streptavidin-protein Afusion protein having an antibody binding site and a biotin bindingsite; (b) an antibody, bound to the antibody binding site, whichantibody is specific for a cell surface protein, and which cell surfaceprotein undergoes endocytosis after binding with the antibody; and (c) abiotinylated prodrug, bound to the biotin binding site.
 10. A complexfor transferring a multidrug resistance (MDR) gene product into a cell,which comprises: (a) a streptavidin-protein A fusion protein having anantibody binding site and a biotin binding site; (b) an antibody, boundto the antibody binding site, which antibody is specific for a cellsurface protein, and which cell surface protein undergoes endocytosisafter binding with the antibody; and (c) a biotinylated MDR geneproduct, bound to the biotin binding site.
 11. A complex fortransferring a single stranded nucleic acid into a cell, whichcomprises: (a) a streptavidin-protein A fusion protein having anantibody binding site and a biotin binding site; (b) an antibody, boundto the antibody binding site, which antibody is specific for a cellsurface protein, and which cell surface protein undergoes endocytosisafter binding with the antibody; and (c) a biotinylated single strandednucleic acid, bound to the biotin binding site.
 12. A complex fortransferring a duplex nucleic acid into a cell, which comprises: (a) astreptavidin-protein A fusion protein having an antibody binding siteand a biotin binding site; (b) an antibody, bound to the antibodybinding site, which antibody is specific for a cell surface protein, andwhich cell surface protein undergoes endocytosis after binding with theantibody; (c) a biotinylated single stranded nucleic acid, having ahomopurine or homopyrimidine portion, bound to the biotin binding site;and (d) a double stranded DNA that forms triplex structure with thebiotinylated single stranded nucleic acid.
 13. A complex fortransferring protein of a pathological bacteria or virus into a cell,which comprises: (a) a streptavidin-protein A fusion protein having anantibody binding site and a biotin binding site; (b) an antibody, boundto the antibody binding site, which antibody is specific for a cellantigen presenting cell surface protein, and which cell surface proteinundergoes endocytosis after binding with the antibody; and (c) abiotinylated protein of a pathological bacteria or virus, bound to thebiotin binding site.
 14. A method for transferring a compound into acell, which comprises the steps of: (a) forming a complex comprising i)a streptavidin-protein A fusion protein having an antibody binding siteand a biotin binding site; ii) an antibody, bound to the antibodybinding site, which antibody is specific for a cell surface protein, andwhich cell surface protein undergoes endocytosis after binding with theantibody; and iii) a biotinylated compound, bound to the biotin bindingsite; (b) isolating the complex from the biotinylated compound that isnot bound to the biotin binding site; and (c) exposing the isolatedcomplex to a cell, so that the compound enters the cell.
 15. The methodof claim 14, wherein the compound is selected from the group consistingof toxin, multidrug resistance (MDR) gene product, single strandednucleic acid, and protein of a pathological bacteria or virus.
 16. Themethod of claim 14, wherein the complex has four antibody binding sitesand four biotin binding sites.
 17. The method of claim 14, whereinstreptavidin component of said streptavidin-protein A fusion protein hasa modified RYD sequence.
 18. The method of claim 14, wherein the complexfurther comprises an antibody that binds transferrin receptor.
 19. Themethod of claim 14, wherein the antibody recognizes a surface antigenselected from the group consisting of: (a) human lymphocyte antigen(HLA-DR); (b) cluster of differentiation (CD33); (c) cluster ofdifferentiation (CD34); and (d) epidermal growth factor (EGF) receptor.20. The method of claim 14, wherein the antibody is an IgG antibody. 21.A method for transferring double stranded DNA into a cell, whichcomprises the steps of: (a) forming a complex comprising: i) astreptavidin-protein A fusion protein having an antibody binding siteand a biotin binding site; ii) an antibody, bound to the antibodybinding site, which antibody is specific for a cell surface protein, andwhich cell surface protein undergoes endocytosis after binding with theantibody; iii) a biotinylated single stranded nucleic acid, bound to thebiotin binding site; and iv) a double stranded DNA that forms triplexstructure with the biotinylated single stranded nucleic acid; and (b)exposing the complex to a cell, so that the double stranded DNA entersthe cell.
 22. The method of claim 21, wherein the complex has fourantibody binding sites and four biotin binding sites.
 23. The method ofclaim 21, wherein streptavidin component of said streptavidin-protein Afusion protein has a modified RYD sequence.
 24. The method of claim 21,wherein the complex further comprises an antibody that binds transferrinreceptor.
 25. The method of claim 21, wherein the antibody recognizes asurface antigen selected from the group consisting of: (a) humanlymphocyte antigen (HLA-DR); (b) cluster of differentiation (CD33); (c)cluster of differentiation (CD34); and (d) epidermal growth factor (EGF)receptor.
 26. The method of claim 21, wherein the antibody is an IgGantibody.